The present exemplary embodiment relates to the use of robust statistical methods in recalibrating measured mass spectra so that measured masses are transformed to be closer to true masses. It finds particular application in conjunction with calibration techniques for mass spectrometry, and will be described with particular reference thereto. However, it is to be appreciated that the present exemplary embodiment is also amenable to other like applications.
The preferred technique for identifying and analyzing biological macromolecules such as peptides and glycans is mass spectrometry (MS). A mass spectrometer is a device able to volatilize/ionize analytes to form vapor-phase ions and determine their absolute or relative molecular masses. A mass spectrometer is generally comprised of at least one ion source, at least one mass analyzer, and at least one detector, though this number of and these particular components are exemplary and not intended to be limiting in any way. Ion sources use methods including, but not limited to, electron bombardment, electrospray ionization, matrix-assisted laser desorption/ionization (MALDI), atmospheric pressure chamber ionization (ACPI), fast atom bombardment, chemical ionization, inductively coupled plasma, and the like or combinations thereof. Mass analyzers use methods including, but not limited to, Fourier transform mass spectrometry (FTMS), quadrupole time-of-flight (QTOF), ion trap, and triple quadrupole. Detectors use methods including, but not limited to, electron multiplication and photon multiplication. Consequently, many permutations of mass spectrometers exist. Those of ordinary skill in the art will recognize these and other types of mass spectrometers, the various methods and theories behind which they operate, and the advantages and disadvantages of the various permutations. The present exemplary embodiment is intended to apply to mass spectra generated by all types of mass spectrometers, with particular application to spectra generated by TOF mass spectrometers. Mass spectrometers may also be hooked up in tandem, the output of the first mass spectrometer being used as the input for the second mass spectrometer; this is known as tandem MS or MS/MS. Further information on mass spectrometry can be found in the Encyclopedia of Chemical Technology, Vol. 15, 4th ed., “Mass Spectrometry”, (1995), and Mass Spectrometry: Principles and Applications, Hoffman et. al., Wiley (1996), the entire contents of both being incorporated by reference herein.
A mass spectrometer measures the mass-to-charge ratio (m/z) of ions. The resulting mass spectrum is plotted as a graph of relative intensity versus m/z. As used herein, a mass spectrum can be the product of either a single mass spectrometer or of a tandem mass spectrometer. It should be noted that the terms peak, m/z, and mass have been used interchangeably in the art even though they do not possess equal dimensional units; however, mass is usually distinguished from m/z. With the mass spectrum and other known data, such as its atomic formula and/or molecular weight, the sample macromolecule can be identified and/or analyzed.
A tandem mass spectrometer fragments ions of selected m/z and measures the m/z of the resulting fragment ions. When the ion is a peptide the fragment is termed a “peptide fragment.” For peptides the most common fragment ions are designated as a-, b-, c-, x-, y-, or z-ions depending on which peptide bond is broken and on which side of the bond the ionic charge is located. When the peptide sequence is written conventionally from N-terminus to C-terminus, a-, b-, and c-ions refer to those fragments including the N-terminus; x-, y-, and z-ions refer to those fragments including the C-terminus. Fragments resulting from a single break in the peptide are called complementary. For example, the b3 ion for the peptide AEFVEVTK is AEF; the complementary y5 ion is VEVTK. a-, b-, c- ions can be distinguished from x-, y-, z-ions by selectively labeling or substituting one end of the peptide, for example by replacing 16O in the terminal acid at the C-terminus with the isotope 18O. This change can be detected and seen in the resulting mass spectrum. In addition, ions are labeled as “parent” ions when they are further fragmented into shorter “daughter” ions.
A mass spectrometer only measures the m/z ratio and hence further work must be done to identify the sample macromolecule. Some factors may be relevant to the identification process. Ionized fragments can further dissociate, for example by losing molecules such as carbon monoxide, water, and ammonia. It should also be noted that isotopes of atoms occur naturally, for example 12C and 13C. These isotopes result in fragments with the same chemical formula but different masses. The various combinations of isotopes possible in an ion create what is called an isotope envelope, where the spacing of peaks and their relative heights due to the natural abundance of isotopes can be predicted. Ions can also be multiply charged; for example, a doubly charged ion can often be recognized by the ½ Dalton (Da) spacing of their accompanying isotope peaks, whereas a triply charged ion can often be recognized by a ⅓ Dalton spacing etc. instead of the normal 1 Dalton spacing for singly charged ions.
The identification of a sample macromolecule depends upon assigning identifications to the peaks in the spectrum. Reliable peak identifications critically depend upon the accuracy of the mass measurement. For example, a peak at m/z 378.83 does not match a b-ion of KHL (mass 379.24) if the measurement uncertainty is plus or minus 0.02, but does match if the measurement uncertainty is 0.5. The larger the measurement uncertainty, the greater the chance of a false assignment and a subsequent failure of identification.
Measurement uncertainty is minimized by accurate estimation of the bias and the precision of the spectrometer. As one of ordinary skill in the art knows, there is a maximum precision inherent to the type of instrument used; for example, FTMS has a precision of 1-2 ppm (0.001 Da at a typical m/z of 1000 Da) and QTOF has a nominal precision of 10 ppm (0.01 Da). However, the precision can also vary depending upon the instrument set-up. The parameters that correct for bias can also vary between set-ups and even between spectra. An example is provided in FIGS. 1 and 2, which show two mass spectra taken at different times on the same TOF instrument from the same sample. The two graphs plot deviation (observed mass minus theoretical mass) versus the measured mass of the peak. In each case the deviation varies linearly with the mass of the peak. A linear fit is shown and it is clear that the bias (the slope of the line) is quite different (note the difference between the y-axes on the two graphs) even though the precision (closeness of points to the line) is similar.
The traditional technique for achieving the theoretical instrument precision is “internal calibration,” meaning the inclusion of a calibrant molecule such as polypropylene glycol in the sample. Several calibrant molecules and methods of their use are known. However, they all suffer from the same drawbacks. First, calibrant molecules give only a limited number of peaks for calibration. Second, there is the chance that calibrant peaks will obscure or diminish signal peaks.
The present exemplary embodiment contemplates a new and improved calibration method, related systems, and media, which overcome the above-referenced problems and others.